Alpha-v Integins and Adenovirus Cell Entry

描述（由申请人提供）：目前，在美国有超过六百万患有心肌缺血的成年人，并且超过六百名人类受试者参与了I/II期临床试验，以评估血管内施用的腺病毒（Ad）载体治疗心血管疾病的安全性。然而，这种临床应用受到来自Ad有效触发宿主先天免疫应答的能力的剂量限制性毒性的阻碍。在之前的资助期间，Scripps研究所Nemerow实验室的研究人员发现，Ad进入浆细胞样树突状细胞通过激活Toll样受体9（TLR 9）诱导1型干扰素和促炎细胞因子的产生。因此，这些研究人员现在处于更好的位置，以确定参与广告介导的先天免疫的信号通路。此外，他们的研究工作发现了一种意想不到的Ad进入宿主细胞的模式。他们证明了Ad颗粒在内体中的部分分解允许释放内部衣壳蛋白，称为pVI，其促进pH非依赖性膜破坏。因此，这些研究人员现在处于一个更好的位置，以扩大知识的广告膜渗透，这是一个过程，是知之甚少的大多数无包膜病毒。 在该提案中，将使用生物化学、生物物理和结构技术的组合来表征pVI的膜溶解特性以及其寡聚体结构和病毒颗粒中的精确位置和方向。从这些研究中产生的信息将被用作产生含有突变pVI蛋白的Ad载体或细胞衍生的纳米颗粒（穹窿）的指导，以确定该衣壳蛋白在内体破坏中的精确作用。最后，细胞培养和体内动物模型将用于研究TLR依赖性和TLR非依赖性先天免疫应答中涉及的Ad进入过程。总之，这些研究应显着增加无包膜病毒如何穿透宿主细胞，以及触发促炎细胞因子的生产的知识。这些信息可以促进更安全和更有效的病毒和非病毒载体的开发。

DESCRIPTION (provided by applicant): Currently, there are over six million adults in the U.S. with myocardial ischemia and more than six hundred human subjects have participated in phase I/II clinical trials to assess the safety of intravascular-administered adenoviral (Ad) vectors to treat cardiovascular diseases. However, such clinical applications are hampered by dose-limiting toxicity stemming from the ability of Ad to potently trigger host innate immune responses. During the previous funding period, researchers in the Nemerow laboratory at The Scripps Research Institute discovered that Ad entry into plasmacytoid dendritic cells induces type one interferon and pro-inflammatory cytokine production via activation of Toll-like receptor nine (TLR9). Thus, these investigators are now in a better position to define the signaling pathways involved in Ad-mediated innate immunity. Moreover, their research efforts uncovered an unanticipated mode of Ad entry into host cells. They demonstrated that partial disassembly of Ad particles in endosomes allows release of an internal capsid protein, designated pVI, which facilitates pH-independent membrane disruption. Thus, these investigators are now in a much better position to extend the knowledge of Ad membrane penetration, a process that is poorly understood for most nonenveloped viruses. In this proposal a combination of biochemical, biophysical, and structural techniques will be used to characterize the membrane lytic properties of pVI as well as its oligomeric structure and precise location and orientation in virus particles. Information arising from these studies will be used as a guide to generate Ad vectors or cell-derived nanoparticles (vaults) containing mutant pVI proteins in order to ascertain the precise role of this capsid protein in endosome disruption. Finally, cell culture and in vivo animal models will be used to investigate the Ad entry processes involved in TLR- dependent and TLR-independent innate immune responses. Together, these studies should significantly increase the knowledge of how a nonenveloped virus penetrates host cells as well as triggers proinflammatory cytokine production. Such information could facilitate the development of safer and more potent viral and non-viral vectors.